Envenomations by the brown recluse spider, Loxosceles reclusa, are a significant source of morbidity in endemic regions of the United States, and misdiagnoses are common. A survey of physicians in the endemic area has shown the economic viability of an accurate diagnostic test for these spider bites. An optimal Loxosceles venom assay entails significant challenges. Unlike the routine construction of enzyme-linked immunosorbent assays (ELISAs) dedicated to the detection of a single protein, this ELISA detects a unique physiologically active protein—sphingomyelinase D (SMD)—abundantly present in a venom containing a myriad of proteins in varying amounts with varying physiological properties. Some of these proteins closely resemble those of other arthropods, making cross-reactivity of proteins a challenge. However, SMD, the major component of venom felt to be responsible for dermal necrosis, has never been reported in any organism other than Loxosceles spiders.
Loxosceles (“slant-legged”) reclusa (“shy and retiring”) (FIG. 1) and other species belonging to the genus Loxosceles are an occasional cause of morbidity and probably a rare cause of mortality in endemic areas (FIG. 2) [Sams01, Cacy99]. Recluse populations become sporadic on either side of the range borders. (From R. Vetter: spiders.ucr.edu with new data in 2002.)
Accurate data on the number of brown recluse envenomations annually is not available. Data collected from poison control centers nationwide show 2,364 brown recluse spider bites (BRSB) reported in 2000, of which 582 had a moderately significant outcome and 21 had a major outcome. [Litovitz01] These data are incomplete and represent a fraction of the total number of probable brown recluse envenomations, which may be estimated by two other methods. Data from our survey of 21 emergency room (ER) physicians and 12 non-ER physicians (FIG. 3) within the central infested area (Missouri, Kansas, Kentucky, Tennessee, Oklahoma, and Arkansas) show an average of 9.62 probable BRSB per year per ER physician. For this emergency room physician population of 3700, a BRSB annual estimate of 35,594 is obtained. For these highly infested areas, emergency room physicians estimate BRSBs comprise 0.4% of the 7.5 million ER visits, or approximately 30,000 probable BRSBs, a similar total. This total includes none of the non-ER visits, which are harder to estimate, and none of the bites out of the central infested area. The total number of possible BRSBs reported by ER physicians is higher, reflected in the total number of test kits needed per year of 17.7 per ER physician or 65,490 tests in the central infested area, an annual market of $1.7 million in central-infested-area ERs alone.
In a review of nineteen documented cases [Sams01a], the most common presenting symptom was pain at the bite site (10 of 19 patients; 53%), which is similar to the frequency of pain in a series including undocumented cases [Cacy99, Gross89]. More common on bites on the extremities, pain begins after two to eight hours and may be severe enough to require narcotics for relief. Pain may be related to sphingomyelinase D degradation of nerve sheath myelin [Clowers96] and may be followed by anesthesia, hypoesthesia, or hyperesthesia [Sams01, Clowers96]. Malaise, fatigue and lightheadedness have been reported in multiple cases, with systemic effects more common in children. Anxiety commonly pervades the first days of Loxosceles envenomation, with dread of severe necrosis or death and worry about a slowly-healing wound (FIGS. 4 and 5). This is a difficult time for patients and their families, and uncertainty in the diagnosis is an additional burden. The most common of the severe systemic effects is hemolytic anemia, both Coombs-positive and Coombs-negative, which can rarely cause lysis of 70% of the red blood cell mass in hours [ouhsc96]. Rarely, disseminated intravascular coagulation may occur [Shenefelt97, Taylor66]. Eight deaths have been recorded in the medical literature as of early 2001, all lacking an accompanying spider for documentation, with most cases in children, generally following severe hemolysis, renal failure and multisystem failure [Sams01].
Many medical conditions cause necrotic wounds and have been misdiagnosed as necrotic arachnidism, leading to a delay in proper treatment. [Vetter98, Stoecker96, Rosenstein87, Vetter03, Oaven99]. These conditions include: anticoagulant necrosis; arthropod bite, e.g., Biting flies, and assassin bugs, kissing bugs, scorpions [Sams01, Vetter98]; atypical mycobacterial infection [Stoecker96]; Bacterial cellulitis; chemical burns; cutaneous vasculitis [Sams01]; eethyma gangrenosum [Sams01]; factitia; Foreign body [Sams01]; Herpes simplex with immunosuppression; Loxoscelism from other species, such as L. deserta, and L. arizonica, L. rufescens [Shenefelt97]; lyme disease [Rosenstein87]; lymphomatoid papulosis [Vetter03]; Necrotic arachnidism: other genera such as egenaria agrestis (Hobo spider), Rabidosa (Lycosa) antelucana, punctulata (wolf spider), Dolomedes scriptus (fishing spider), peucetia viridans (green lynx spider), Chemacanthium mildei (sac spider) [Sams01]; necrotizing fasciitis [Maisel94]; pyoderma gangrenosum as seen in FIG. 6 (pyoderma gangrenosum can be distinguished from necrotic arachnidism in cases with multiple inflammatory pustules or dusky, volcano-like hemorrhagic nodules, but a single ulcer can be suggestive of a spider bite and Loxosceles envenomations can be followed by pyoderma gangrenosum [Sams01, Hoover90]; syphilitic chancre [Cacy99]; Sweet's syndrome Sams01]; sporotrichosis [Oaven99]; tularemia [Stoecker96]; and ulcers, both diabetic and stasis [Shenefelt97].
Streptococci may be taken as the prototype for a number of bacteria that can cause necrosis. These include Clostridium difficile, Vibrio vulnificus, and Pseudomonas aeruginosa, (as well as other Gram-negative rods that cause eethyma gangrenosum). Extensive bacterial cellulitis, especially when the lesion is progressing in size and swollen diffusely, needs specific antibiotic and surgical management. In the news in 2002, there was a case of anthrax in a child in New York City, initially misdiagnosed as a spider bite. Cases of tularemia, atypical mycobacterial infections, and even multiple cases of lymphomatoid papulosis [Vetter03] have been initially misdiagnosed as spider bites. Mistaking these lesions for spider bites can have adverse consequences for the patient.
As yet, without clinical tests for loxoscelism, for cases in which the spider has not been recovered, the wound is diagnosed based upon the presence of typical morphology, a compatible history (such as a bite following putting on clothing after long storage), and whether the bite occurred within the expected territorial range. [Sams01] As an example, the wound in FIG. 7, appearing in an email from a physician to one of the inventors, coming from an area of the country with no documented cases of loxoscelism, may well have been due to trauma or abuse rather than to Loxosceles envenomation, but we could not be certain. Unfortunately, in this case as in other such cases, the potential for litigation is present, and if this occurs, litigation outcome may depend upon an uncertain diagnosis.
A sensitive and specific clinical test has been sought for envenomations with Loxosceles reclusa. A passive hemagglutination inhibition (PHAI) test was reported to be successful in identifying Loxosceles reclusa experimental envenomations in guinea pigs, with 90% sensitivity up to three days after venom injection, and 100% specificity as far as false identification of other spider species, but the test is difficult to perform [Sams01, Barrett93]. A lymphocyte transformation test has also been developed, but is rarely used because of expense and delayed appearance of a positive test result [Berger73]. Proteins contained in Loxosceles venom are immunogenic with significant titers of anti-Loxosceles IgG antibody formation when venom is inoculated multiple times in the rabbit model [Gomez99]. However, antibody response in humans, across Loxosceles species, appears to be weak. Only four of 20 patients bitten with L. gaucho and treated with serum therapy had antibodies to L. gaucho venom [Barbaro92]. In another study, there were no antibodies to Loxosceles venom in measurements taken out to 30 days [Guilherme01]. Thus several experimental methods have been developed to detect the presence of Loxosceles venom, but none are simple enough to be commercially available for confirmation of envenomation in patients with suspected Loxosceles-induced lesions.
Immunoassay methods and devices comprising swabs used for analyzing samples are known to the art. U.S. Patent Publication No. 2005/0136553 discloses a device in which a swab is contacted by a fluid contained in a fluid chamber via a flow channel, and also containing an assay in fluid communication with the swab, the fluid chamber and the flow channel. U.S. Pat. No. 6,248,294 describes a substantially self-contained diagnostic test for collecting and analyzing a biological specimen having a tubular housing defining a specimen chamber for receiving a biological specimen collected from a swab. U.S. Pat. No. 4,582,699 discloses a kit for detection of gonorrhea in which an inert strip with antibody to the antibody to be detected immobilized thereon is inserted into the sample and subsequently exposed to a reagent for detecting binding. U.S. Pat. No. 4,916,057 describes an immunoassay procedure for detection of Chlamydia trachomatis antigen in a sample collected on a swab comprising extracting the sample from the swab with a basic solution that is subsequently neutralized before conducting the immunoassay. U.S. Pat. Nos. 5,753,262 and 4,943,522 disclose lateral flow immunoassays used as pregnancy tests. U.S. Pat. No. 5,163,441 describes a swab for collecting microbiological cultures comprising a swabbing tip made with a non-toxic polyurethane foam having open cells at its exposed surface. U.S. Pat. No. 5,084,245 discloses a device and method involving expressing liquid from the swab for analysis.
All patents and publications referred to herein are incorporated by reference to the extent not inconsistent herewith for the purpose of providing written description and enablement of art-known aspects of this invention.